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目的:纵向分析自然免疫压力下慢性1b型HCV感染者E2基因的系统进化模式及正选择位点,初步探讨E2基因正选择位点的分布与已知功能区之间的关系。方法:未抗病毒治疗的3例慢性1b型HCV感染者及其系列血清样本均来自中国人HCV感染患者系列标本库(CQueen cohort)。对每例感染者3个连续时间点的血清样本进行HCV5′端6000bp基因组的扩增、克隆,每个时间点随机挑选25～33个克隆对E2基因测序,用MEGA软件构建系统进化树,用固定效应似然法检测HCV E2基因的正选择位点。结果:3例HCV慢性感染者的9份血清样本均获清晰、无杂带的6030bp的目的片段。慢性HCV感染者体内病毒准种的复杂程度并非随着感染时间的延长而进行性增加,在某个时间点上准种变异株的序列可能会趋于一致。准种优势变异株在间隔半年时间以上已发生更替。对每位患者动态的准种变异株进行正选择位点的分析,共检测到5个氨基酸(aa)位点:aa384、aa399、aa410、aa475和aa522,分布于HCV E2基因HVR1区、HVR2区及HCV E2-CD81分子结合区。结论:基于动态变化的HCV准种优势株的相关研究,可考虑选择间隔半年以上时间点的血清样本作为研究对象。慢性HCV感染过程中,HCV E2基因位于重要功能区的aa位点受到了宿主强烈的免疫压力,aa384、aa399及aa410位点的变异可能导致HCV发生体液免疫逃逸;而aa475和aa522位点的变异则可能通过影响HVR2区的功能或HCV E2-CD81分子的亲和力或改变CD81分子的结构而导致HCV发生先天免疫逃逸。该研究为分析HCV C-NS3基因片段的正选择位点奠定了良好的基础。
OBJECTIVE: To analyze systematically the evolutionary pattern and positive selection sites of E2 gene in chronic type 1b HCV infected with innate immune pressure, and to explore the relationship between the distribution of E2 gene positive selection sites and known functional areas. Methods: Three patients without systemic antiviral therapy for chronic type 1b HCV infection and their serogroups were all from a Chinese population of HCV infected patients with CQueen cohort. The HCV genome was amplified by PCR at the 5 ’end of each HCV infection sample from 3 consecutive time points. The E2 gene was randomly selected from 25 to 33 clones at each time point. The phylogenetic tree was constructed using MEGA software. Fixed Effect Likelihood Detection of Positive Selection Site of HCV E2 Gene. Results: All the 9 serum samples from 3 chronic HCV infected patients were clear and unobserved 6030bp. The complexity of the viral quasispecies in chronic HCV-infected individuals does not increase progressively with the duration of the infection, and the sequence of the quasispec is likely to converge at some point in time. The dominant species of mutants have been replaced at intervals of more than six months. Five candidate amino acid (aa) sites were detected in each of the quasispecies: aa384, aa399, aa410, aa475 and aa522, which were located in the HVR1 and HVR2 regions of HCV E2 And HCV E2-CD81 molecular binding region. CONCLUSIONS: Based on the dynamic changes of HCV quasispecies, serum samples taken at intervals of more than six months may be considered as the study subjects. During chronic HCV infection, HCV E2 gene is strongly immunized by host in aa site of important functional domain. Mutation at aa384, aa399 and aa410 sites may result in humoral immune escape of HCV. However, mutations in aa475 and aa522 sites It is possible to cause innate immune escape of HCV by affecting the function of the HVR2 region or the affinity of the HCV E2-CD81 molecule or altering the structure of the CD81 molecule. This study laid a good foundation for analyzing the positive selection site of HCV C-NS3 gene fragment.